Chick Cranial Neural Crest Cells Research Articles

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

Chick cranial neural crest cells release extracellular vesicles that are critical for their migration.

ABSTRACTThe content and activity of extracellular vesicles purified from cell culture media or bodily fluids have been studied extensively; however, the physiological relevance of exosomes within normal biological systems is poorly characterized, particularly during development. Although exosomes released by invasive metastatic cells alter migration of neighboring cells in culture, it is unclear whether cancer cells misappropriate exosomes released by healthy differentiated cells or reactivate dormant developmental programs that include exosome cell–cell communication. Using chick cranial neural fold cultures, we show that migratory neural crest cells, a developmentally critical cell type and model for metastasis, release and deposit CD63-positive 30–100 nm particles into the extracellular environment. Neural crest cells contain ceramide-rich multivesicular bodies and produce larger vesicles positive for migrasome markers as well. We conclude that neural crest cells produce extracellular vesicles including exosomes and migrasomes. When Rab27a plasma membrane docking is inhibited, neural crest cells become less polarized and rounded, leading to a loss of directional migration and reduced speed. These results indicate that neural crest cell exosome release is critical for migration.

Connexin 43 Regulates Multiple Aspects of Chick Cranial Neural Crest Cell Development

Connexins are transmembrane proteins that cluster to form intercellular channels called gap junctions, which allow direct diffusion of small molecules and ions between adjacent cells. Connexin 43 (Cx43) is one of the predominant components of gap junctions and is expressed in multiple organisms and their tissues. To date, however, very little is known about the role of gap junctions, and specifically Cx43, in the neural crest. Our prior immunohistochemical analyses revealed that Cx43 is expressed in premigratory cranial neural crest cells both prior to and during their epithelial‐to‐mesenchymal transition (EMT), and in migratory neural crest cells. Given the robust expression of Cx43 in premigratory neural crest cells, we sought to elucidate the role of Cx43, and gap junctions in particular, during neural crest cell EMT. To this end, we evaluated the presence of gap junctions in the neural crest and performed perturbation experiments to alter the levels of Cx43 in both premigratory and migratory neural crest cells. Our fixed and live imaging results reveal that gap junction formation occurs within both the premigratory and migratory neural crest cell populations. Moreover, morpholino‐mediated reduction in Cx43 protein levels prevents neural crest cells from forming functional gap junctions, as assessed by live imaging of transfer of the gap junction‐specific dye Calcein in the embryo. Additionally, depletion of Cx43 from premigratory neural crest cells reduces the size of the premigratory neural crest cell domain and also delays the emergence of neural crest cells from the dorsal neural tube. This delayed neural tube exit phenotype can be rescued by introduction of full‐length rat Cx43 that is refractory to the morpholino. These data suggest that gap junctions, and Cx43 in particular, are involved in early neural crest cell specification and in the emergence of neural crest cells from the neural tube.Support or Funding InformationThe work was supported by an American Association for Anatomy Postdoctoral Fellowship to K.J. and grants to L.A.T. (NIH R01DE024217, American Cancer Society RSG‐15‐023‐01‐CSM).

The gap junction protein connexin 43 controls multiple aspects of cranial neural crest cell development.

Gap junctions are intercellular channels between cells that facilitate cell-cell communication. Connexin 43 (Cx43; also known as GJA1), the predominant gap junction protein in vertebrates, is expressed in premigratory cranial neural crest cells and is maintained throughout the neural crest cell epithelial-to-mesenchymal transition (EMT), but its function in these cells is unknown. To this end, we used a combination of in vivo and ex vivo experiments to assess gap junction formation, and Cx43 function, in chick cranial neural crest cells. Our results demonstrate that gap junctions exist between premigratory and migratory cranial neural crest cells and depend on Cx43 for their function. In the embryo, Cx43 knockdown just prior to EMT delays the emergence of Cx43-depleted neural crest cells from the neural tube, but these cells eventually successfully emigrate and join the migratory stream. This delay can be rescued by introduction of full-length Cx43 into Cx43-depleted cells. Furthermore, Cx43 depletion reduces the size of the premigratory neural crest cell domain through an early effect on neural crest cell specification. Collectively, these data identify new roles for Cx43 in chick cranial neural crest cell development.

Alcohol-mediated calcium signals dysregulate pro-survival Snai2/PUMA/Bcl2 networks to promote p53-mediated apoptosis in avian neural crest progenitors.

Prenatal alcohol exposure causes distinctive craniofacial anomalies that arise, in part, from the apoptotic elimination of neural crest (NC) progenitors that form the face. This vulnerability of NC to alcohol is puzzling as they normally express the transcriptional repressor Snail1/2 (in chick Snai2), which suppresses apoptosis and promotes their migration. Here, we investigate alcohol's impact upon Snai2 function. Chick cranial NC cells were treated with acute alcohol (52 mM, 2 hr). We evaluated NC migration, gene expression, proliferation, and apoptosis thereafter. Transient alcohol exposure induced Snai2 (191% ± 23%; p = .003) and stimulated NC migration (p = .0092). An alcohol-induced calcium transient mediated this Snai2 induction, and BAPTA-AM blocked whereas ionomycin mimicked these pro-migratory effects. Alcohol suppressed CyclinD1 protein content (59.1 ± 12%, p = .007) and NC proliferation (19.7 ± 5.8%, p < .001), but these Snai2-enriched cells still apoptosed in response to alcohol. This was explained because alcohol induced p53 (198 ± 29%, p = .023), and the p53 antagonist pifithrin-α prevented their apoptosis. Moreover, alcohol counteracted Snai2's pro-survival signals, and Bcl2 was repressed (68.5 ± 6.0% of controls, p = .016) and PUMA was not induced, while ATM (1.32-fold, p = .01) and PTEN (1.30-fold, p = .028) were elevated. Alcohol's calcium transient uncouples the Snai2/p53 regulatory loop that normally prevents apoptosis during EMT. This represents a novel pathway in alcohol's neurotoxicity, and complements demonstrations that alcohol suppresses PUMA in mouse NC. We propose that the NCs migratory behavior, and their requirement for Snai2/p53 co-expression, makes them vulnerable to stressors that dysregulate Snai2/p53 interactions, such as alcohol.

Cadherin-6B proteolytic N-terminal fragments promote chick cranial neural crest cell delamination by regulating extracellular matrix degradation

During epithelial-to-mesenchymal transitions (EMTs), chick cranial neural crest cells simultaneously delaminate from the basement membrane and segregate from the epithelia, in part, via multiple protease-mediated mechanisms. Proteolytic processing of Cadherin-6B (Cad6B) in premigratory cranial neural crest cells by metalloproteinases not only disassembles cadherin-based junctions but also generates shed Cad6B ectodomains or N-terminal fragments (NTFs) that may possess additional roles. Here we report that Cad6B NTFs promote delamination by enhancing local extracellular proteolytic activity around neural crest cells undergoing EMT en masse. During EMT, Cad6B NTFs of varying molecular weights are observed, indicating that Cad6B may be cleaved at different sites by A Disintegrin and Metalloproteinases (ADAMs) 10 and 19 as well as by other matrix metalloproteinases (MMPs). To investigate Cad6B NTF function, we first generated NTF constructs that express recombinant NTFs with similar relative mobilities to those NTFs shed in vivo. Overexpression of either long or short Cad6B NTFs in premigratory neural crest cells reduces laminin and fibronectin levels within the basement membrane, which then facilitates precocious neural crest cell delamination. Zymography assays performed with supernatants of neural crest cell explants overexpressing Cad6B long NTFs demonstrate increased MMP2 activity versus controls, suggesting that Cad6B NTFs promote delamination through a mechanism involving MMP2. Interestingly, this increase in MMP2 does not involve up-regulation of MMP2 or its regulators at the transcriptional level but instead may be attributed to a physical interaction between shed Cad6B NTFs and MMP2. Taken together, these results highlight a new function for Cad6B NTFs and provide insight into how cadherins regulate cellular delamination during normal developmental EMTs as well as aberrant EMTs that underlie human disease.

Chick cranial neural crest cells use progressive polarity refinement, not contact inhibition of locomotion, to guide their migration

To move directionally, cells can bias the generation of protrusions or select among randomly generated protrusions. Here we use 3D two-photon imaging of chick branchial arch 2 directed neural crest cells to probe how these mechanisms contribute to directed movement, whether a subset or the majority of cells polarize during movement, and how the different classes of protrusions relate to one another. We find that, in contrast to Xenopus, cells throughout the stream are morphologically polarized along the direction of overall stream movement and do not exhibit contact inhibition of locomotion. Instead chick neural crest cells display a progressive sharpening of the morphological polarity program. Neural crest cells have weak spatial biases in filopodia generation and lifetime. Local bursts of filopodial generation precede the generation of larger protrusions. These larger protrusions are more spatially biased than the filopodia, and the subset of protrusions that are productive for motility are the most polarized of all. Orientation rather than position is the best correlate of the protrusions that are selected for cell guidance. This progressive polarity refinement strategy may enable neural crest cells to efficiently explore their environment and migrate accurately in the face of noisy guidance cues.

FoxD3 regulates cranial neural crest EMT via downregulation of tetraspanin18 independent of its functions during neural crest formation

The scaffolding protein tetraspanin18 (Tspan18) maintains epithelial cadherin-6B (Cad6B) to antagonize chick cranial neural crest epithelial-to-mesenchymal transition (EMT). For migration to take place, Tspan18 must be downregulated. Here, we characterize the role of the winged-helix transcription factor FoxD3 in the control of Tspan18 expression. Although we previously found that Tspan18 mRNA persists several hours past the stage it would normally be downregulated in FoxD3-deficient neural folds, we now show that Tspan18 expression eventually declines. This indicates that while FoxD3 is crucial for initial downregulation of Tspan18, other factors subsequently impact Tspan18 expression. Remarkably, the classical EMT transcription factor Snail2 is not one of these factors. As in other vertebrates, FoxD3 is required for chick cranial neural crest specification and migration, however, FoxD3 has surprisingly little impact on chick cranial neural crest cell survival. Strikingly, Tspan18 knockdown rescues FoxD3-dependent neural crest migration defects, although neural crest specification is still deficient. This indicates that FoxD3 promotes cranial neural crest EMT by eliciting Tspan18 downregulation separable from its Tspan18-independent activity during neural crest specification and survival.

Cadherin-6B is proteolytically processed during epithelial-to-mesenchymal transitions of the cranial neural crest

The epithelial-to-mesenchymal transition (EMT) is a highly coordinated process underlying both development and disease. Premigratory neural crest cells undergo EMT, migrate away from the neural tube, and differentiate into diverse cell types during vertebrate embryogenesis. Adherens junction disassembly within premigratory neural crest cells is one component of EMT and, in chick cranial neural crest cells, involves cadherin-6B (Cad6B) down-regulation. Whereas Cad6B transcription is repressed by Snail2, the rapid loss of Cad6B protein during EMT is suggestive of posttranslational mechanisms that promote Cad6B turnover. For the first time in vivo, we demonstrate Cad6B proteolysis during neural crest cell EMT, which generates a Cad6B N-terminal fragment (NTF) and two C-terminal fragments (CTF1/2). Coexpression of relevant proteases with Cad6B in vitro shows that a disintegrin and metalloproteinases (ADAMs) ADAM10 and ADAM19, together with γ-secretase, cleave Cad6B to produce the NTF and CTFs previously observed in vivo. Of importance, both ADAMs and γ-secretase are expressed in the appropriate spatiotemporal pattern in vivo to proteolytically process Cad6B. Overexpression or depletion of either ADAM within premigratory neural crest cells prematurely reduces or maintains Cad6B, respectively. Collectively these results suggest a dual mechanism for Cad6B proteolysis involving two ADAMs, along with γ-secretase, during cranial neural crest cell EMT.

In vivo analysis reveals a critical role for neuropilin-1 in cranial neural crest cell migration in chick

The neural crest provides an excellent model system to study invasive cell migration, however it is still unclear how molecular mechanisms direct cells to precise targets in a programmed manner. We investigate the role of a potential guidance factor, neuropilin-1, and use functional knockdown assays, tissue transplantation and in vivo confocal time-lapse imaging to analyze changes in chick cranial neural crest cell migratory patterns. When neuropilin-1 function is knocked down in ovo, neural crest cells fail to fully invade the branchial arches, especially the 2nd branchial arch. Time-lapse imaging shows that neuropilin-1 siRNA transfected neural crest cells stop and collapse filopodia at the 2nd branchial arch entrances, but do not die. This phenotype is cell autonomous. To test the influence of population pressure and local environmental cues in driving neural crest cells to the branchial arches, we isochronically transplanted small subpopulations of DiI-labeled neural crest cells into host embryos ablated of neighboring, premigratory neural crest cells. Time-lapse confocal analysis reveals that the transplanted cells migrate in narrow, directed streams. Interestingly, with the reduction of neuropilin-1 function, neural crest cells still form segmental migratory streams, suggesting that initial neural crest cell migration and invasion of the branchial arches are separable processes.

In vivo analysis reveals a critical role for neuropilin-1 in chick cranial neural crest cell migration

In vivo analysis reveals a critical role for neuropilin-1 in chick cranial neural crest cell migration

Expression of an engrailed‐like gene during development of the early embryonic chick nervous system

The engrailed gene has been identified in Drosophila as an important developmental gene involved in the control of segmentation. Here we describe the embryonic expression of a chicken gene, ChickEn (Darnell et al.: J Cell Biol 103(5):311a, 1986), which contains homology to the Drosophila engrailed gene. Northern blots of early chick embryo tissue poly(A)+ RNA resulted in hybridization to at least three bands expressed predominantly in the brain/head region when probed with ChickEn genomic fragments. Eight cDNA clones generated from embryonic day 6 (stage 29-30) chick brain poly(A)+ RNA are identical in their nucleotide sequence with the ChickEn genomic clone. In situ hybridization to sections of 4-day (stage 24) embryos indicated that ChickEn transcripts were concentrated in the posterior mesencephalon and anterior metencephalon. In cultures of chick cranial neural crest cells (eight to nine somites; stage 9) ChickEn transcripts were localized in a subset (approx. 8%) of cells examined after 2 days in culture. A mouse monoclonal antibody, inv-4D9D4, made by Coleman and Kornberg recognizes the engrailed-like homeo domain of the engrailed and invected proteins (Martin-Blanco, Coleman, and Kornberg, personal communication). Patel, Coleman, Kornberg and Goodman (unpublished) have shown that this antibody binds to the hindbrain of 2-day-old chick embryos. We have confirmed these results and shown that this antibody binds to the same region of 4-day (stage 24) chick brains that in situ hybridization showed contained ChickEn transcripts. This antibody also recognizes a homeo domain-containing ChickEn peptide expressed as a beta-galactosidase fusion protein in Drosophila cell culture. We have not detected ChickEn protein in any tissue prior to eight to nine somites (stage 9). These results delineate the major expression pattern of the ChickEn gene during early (prior to stage 30) embryonic development in the chick.

Development of choline acetyltransferase activity in chick cranial neural crest cells in culture

Cultures of chick cranial neural crest cells, when grown in medium containing horse serum (HS), underwent morphological changes leading to the formation, after 6 days in culture, of dense aggregates of cells, some of which produced processes resembling neurite fascicles. In contrast, cranial neural crest cells grown under identical conditions, but with fetal calf serum (FCS) instead of HS, did not form aggregates; after 7–8 days, most of these cells were heavily pigmented. Choline acetyltransferase (CAT) activity was absent or low in freshly dissected neural tubes from embryos of stage 9 and in neural tubes in culture for 2 days in medium containing either HS or FCS. CAT activity was also absent in neural crest cells which had grown out in the presence of FCS for 6 days or in the presence of HS for 2 or 4 days. Cells grown in medium containing HS for 6 days developed significant levels of CAT activity. These results demonstrate that cranial neural crest cells in culture have the capacity to differentiate into cholinergic cells under specific conditions. When neural crest cells which had migrated from neural tubes in medium containing HS were shifted to medium containing FCS for 4 days, no CAT activity was detected. However, when neural crest cells which had migrated in medium containing FCS were shifted to medium containing HS for 4 days, the cells formed aggregates and CAT activity was present. This demonstrates that the kind of serum present in the medium did not select for the outgrowth of a specific predetermined population of neural crest cells from the neural tube, but rather that some factor associated with subsequent culture conditions influenced the cells to become cholinergic cells or pigmented cells.